Putative transcription factor Nrg1 is involved in the hyphal branching and the cell wall structure formation in the filamentous fungus Pleurotus ostreatus

Nrg1 is a C2H2 zinc finger transcription factor that functions in various cellular processes related to environmental responses and cell wall synthesis in yeast. The present study investigated the function of Nrg1 in filamentous development in the white-rot fungus Pleurotus ostreatus for the first time. The Δnrg1 strains exhibited a higher frequency and larger angles of hyphal branching than the wild-type strain, suggesting nrg1 is essential to hyphal branching regulation. Transmission electron microscopy observations indicated no significant change in cell wall thickness; however, the black layer on the cell wall surface, thought to be composed of proteins, including hydrophobins, was reduced. The reduced hydrophobin gene expression and hyphal hydrophobicity corroborate these observations. The nrg1 disruption resulted in decreased levels of α-glucan and β-glucan content in the cell wall and the expression levels of their synthase genes. Fluorescent microscopy using protein probes for cell wall surface polysaccharides also suggested that in the Δnrg1 strains, there was an abnormal localization of chitin and β-glucan. In contrast, the fluorescent signals due to α-glucan and mannan were increased. These data demonstrate that Nrg1 is involved in the cell wall surface and structure formation via the gene expression of hydrophobin and polysaccharide synthases. This study provides the first evidence of the role of Nrg1 in filamentous fungi, especially in hyphal branching. It provides a clue toward a comprehensive understanding of the mechanisms regulating cell wall synthesis in white-rot fungi.